Blood platelets prevent hemorrhage, mediate diverse pathologic processes, including inflammation and thrombosis, and are frequently reduced in toxic response to cancer therapy. The central role of platelets in common disorders makes it important to know how megakaryocytes (MKs) terminate differentiation and release platelets into the circulation. Microtubule-based MK cytoplasmic extensions known as proplatelets serve as the immediate precursors of blood platelets and their elaboration requires the activities of transcription factors GATA1 and NF-E2. However, most underlying structural and signaling pathways and relevant targets of the critical transcriptional regulators remain unknown. The platelet-specific beta-tubulin isoform beta1 is a major component of proplatelet and platelet marginal microtubules and requires NF-E2 for expression. Beta1 tubulin-/- mice produce few and spherocytic platelets and thus attest to the importance of this lineage-restricted cytoskeletal factor in thrombopoiesis. We have isolated several MK-expressed proteins that interact with the divergent C-terminus of beta1 tubulin and their initial characterization leads to specific hypotheses about their functions in platelet synthesis or function. Additionally, in an effort to generate a cogent molecular classification of individual stages in MK maturation, we have captured gene expression profiles from MK subpopulations isolated using flow cytometry. The preliminary results again suggest roles for selected signaling pathways that may be tested in primary mouse MKs. The Specific Aims of this proposal are: (1) To characterize the associations and functions of beta1 tubulin-interacting MK proteins, especially a novel Ran-binding factor RanBPM-2 and the secretory leukocyte protease inhibitor SLPI. (2) To identify the major molecular events corresponding to recognized morphologic and functional transitions in MK maturation and the molecular pathways that are active at different stages in thrombopoiesis. (3) To test the functions of a limited number of genes identified through Aims 1 and 2 in aspects of platelet biogenesis. We are using an effective and flexible strategy to over express genes in cultured primary MKs, followed by functional and structural examination of the consequences. To facilitate the Aims of this project, we also propose to adapt the method to abrogate expression of endogenous genes. We expect the collective results of our studies to advance current understanding of how MKs produce blood platelets.